Published OnlineFirst March 12, 2019; DOI: 10.1158/0008-5472.CAN-18-2849 Cancer Metabolism and Chemical Biology Research Free Fatty Acids Rewire Cancer Metabolism in Obesity-Associated Breast Cancer via Estrogen Receptor and mTOR Signaling Zeynep Madak-Erdogan1,2,3,4,5,6, Shoham Band1,YiruC.Zhao1, Brandi P. Smith1, Eylem Kulkoyluoglu-Cotul1, Qianying Zuo1, Ashlie Santaliz Casiano2, Kinga Wrobel1, Gianluigi Rossi7, Rebecca L. Smith7, Sung Hoon Kim8, John A. Katzenellenbogen8, Mariah L. Johnson9, Meera Patel9, Natascia Marino9,10, Anna Maria V. Storniolo9,10, and Jodi A. Flaws11 Abstract Obesity is a risk factor for postmenopausal estrogen recep- naling, was able to block changes induced by FFA and was þ tor alpha (ERa)-positive (ER ) breast cancer. Molecular more effective in the presence of FFA. Collectively, these data mechanisms underlying factors from plasma that contribute suggest a role for obesity-associated gene and metabolic rewir- þ to this risk and how these mechanisms affect ERa signaling ing in providing new targetable vulnerabilities for ER breast have yet to be elucidated. To identify such mechanisms, we cancer in postmenopausal women. Furthermore, they provide performed whole metabolite and protein profiling in plasma a basis for preclinical and clinical trials where the impact of samples from women at high risk for breast cancer, which led agents that target ERa and mTOR signaling cross-talk would be þ us to focus on factors that were differentially present in plasma tested to prevent ER breast cancers in obese postmenopausal of obese versus nonobese postmenopausal women. These women. studies, combined with in vitro assays, identified free fatty acids (FFA) as circulating plasma factors that correlated with Significance: These findings show that obesity-associated þ increased proliferation and aggressiveness in ER breast cancer changes in certain blood metabolites rewire metabolic pro- cells. FFAs activated both the ERa and mTOR pathways and grams in cancer cells, influence mammary epithelial cell rewired metabolism in breast cancer cells. Pathway preferen- tumorigenicity and aggressiveness, and increase breast cancer tial estrogen-1 (PaPE-1), which targets ERa and mTOR sig- risk. Introduction Sedentary lifestyle and western-style, fat- and sugar-rich diets, combined with low estrogen levels in postmenopausal women, The rate of increase in BMI is higher for women in the United aggravate this problem, making postmenopausal women more States and percent of cancer cases attributable to excess body susceptible to weight gain, fat redistribution to abdominal weight is twice as high for women compared with men (1, 2). areas, dyslipidemia, hypertension, and insulin resistance, which are the major hallmarks of metabolic syndrome (3). In 1Department of Food Sciences and Human Nutrition, University of Illinois, fact, almost 70% of postmenopausal women in the United Urbana-Champaign, Urbana, Illinois. 2Division of Nutritional Sciences, University States are overweight or obese. A weight gain of 55 or more of Illinois, Urbana-Champaign, Urbana, Illinois. 3National Center for Supercom- pounds from age 18 increases breast cancer risk by 50% (4). þ puting Applications, University of Illinois, Urbana-Champaign, Urbana, Illinois. Being overweight after menopause increases ER breast cancer 4Cancer Center at Illinois, University of Illinois, Urbana-Champaign, Urbana, – 5 risk by 70% (5 7). Eighty-two different studies including Illinois. Beckman Institute for Advanced Science and Technology, University of analysis of more than 200,000 women with breast cancer Illinois at Urbana-Champaign, Urbana, Illinois. 6Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois. showed that obesity increases mortality both in premenopausal 7Department of Pathobiology, University of Illinois, Urbana-Champaign, Urbana, and postmenopausal women (8). Obesity-associated cancers Illinois. 8Department of Chemistry, University of Illinois, Urbana-Champaign, are a significant clinical problem and uncovering and targeting Urbana, Illinois. 9Susan G. Komen Tissue Bank at the IU Simon Cancer Center, obesity-associated molecules and signaling pathways activated Indianapolis, Indiana. 10Department of Medicine, Indiana University School of 11 by these molecules may identify populations at risk and reduce Medicine, Indianapolis, Indiana. Department of Comparative Biosciences, Uni- breast cancer–related deaths. versity of Illinois, Urbana-Champaign, Urbana, Illinois. Systemic hyperlipidemia, hyperglycemia, insulin resistance, Note: Supplementary data for this article are available at Cancer Research increased estrogen production by adipose tissue, and increased Online (http://cancerres.aacrjournals.org/). inflammation associated with obesity contribute to the risk and Corresponding Author: Zeynep Madak-Erdogan, University of Illinois at Urbana development of breast cancer (9). Obesity-associated factors Champaign, 359 ERML Hall, 1201 W. Gregory Dr., Urbana, IL 61801-3704. such as IGF1, adipokines, and cytokines modulate oncogenic Phone: 217-300-9063; Fax: 217-265-0925; E-mail: [email protected] PI3K and mTOR signaling pathways (10). Tumor-associated doi: 10.1158/0008-5472.CAN-18-2849 cholesterol metabolites, such as 27-hydroxycholesterol, also Ó2019 American Association for Cancer Research. increased risk of breast cancer metastasis and worsen breast 2494 Cancer Res; 79(10) May 15, 2019 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 12, 2019; DOI: 10.1158/0008-5472.CAN-18-2849 ERa in Obesity-Associated Breast Cancer cancer outcomes (11–13). Yet, we still lack the information controls) with respect to the distribution of the abovemen- regarding other molecules from plasma that increase the breast tioned confounders in susceptible group. cancer risk. All studies were approved by the University of Illinois, Urbana- Our aim in this study was to identify and test the impact of Champaign Institutional Review Board (UIUC IRB protocol various circulating factors in blood associated with breast cancer number 06741). The Midlife Women's Health Study is a longi- risk. Our hypothesis was that specific circulating metabolites and tudinal study on risk factors for hot flashes in women who are þ proteins, detectable in plasma, increase the risk of ER breast residents of the Baltimore metropolitan region, which includes cancer in obese postmenopausal women compared with their Baltimore city and several of its surrounding counties (15). This nonobese counterparts. Using a multiple -omics approach, we parent study, named the Midlife Women's Health Study, was identified certain free fatty acids (FFA) that are relevant to obesity- specifically designed to test the hypothesis that obesity is associ- associated breast cancer risk and uncovered ERa and mTOR ated with hot flashes through mechanisms that involve early pathway–dependent metabolic rewiring in breast cancer cells ovarian failure, altered estradiol levels, and selected genetic poly- under conditions that mimic plasma from obese postmenopausal morphisms in steroidogenic enzymes and steroid hormone recep- women. We have previously described the identification and tors. We analyzed plasma samples from 37 nonobese and 63 design of novel pathway preferential estrogens that modulate obese postmenopausal women who were two to three years into ERa and mTOR signaling cross-talk (14). One of these com- menopause at the time of sampling. In addition, we analyzed pounds, termed pathway preferential estrogen 1 (PaPE-1), was plasma samples from 21 postmenopausal women who were initially identified in a screen of estrogen-like molecules that had obese at the initiation of the study and later lost weight. lower affinity for the estrogen receptor, and stimulated extranu- clear ER activity with limited effects on nuclear ER-target gene Plasma preparation expression. PaPE-1 modulated extranuclear ER-initiated kinase Blood is drawn into the Plasma Separator tube (Vacutainer signaling, particularly mTOR pathway, without inducing ERa Venous Blood Collection Tubes: SSTÃ Plasma Separation Tube, recruitment to chromatin. In in vivo experiments, PaPE-1 treat- Thermo Fisher Scientific, catalog no. 0268396) and gently mixed ment reduced ovariectomy-induced weight gain, blood triglycer- by inverting the tube five times. Forty-five minutes (Æ10 minutes) ide levels, and fat deposition in an ERa-dependent manner, after the blood has been drawn, the Plasma Separator Tube is without stimulating the uterus or mammary gland, and displayed placed into a minicentrifuge (Eppendorf centrifuge 5702) and a pattern of metabolic tissue-selective activity that would be centrifuged at 1,200 rcf for 10 minutes at room temperature. A optimal for preventing breast cancer in postmenopausal women repeater pipet is used to aliquot 600 mL of the plasma into each of by reducing fat accumulation in the body (14). In this study, five cryogenic vials. Samples are stored at À80C until use. PaPE-1, was able to block FFA-dependent changes in human breast cancer cells. Hence, we have uncovered a novel role for Primary cell culture extranuclear-initiated ERa signaling in rewiring breast cancer cell Primary mammary epithelial cells were isolated from 10 cryo- metabolism in response to obesity-associated factors in the plas- preserved breast biopsies of healthy donors as described previ- ma. Our findings provide a basis for preventing or inhibiting ously